Bcl-2 Transcription Research Articles

Survival advantage of native and engineered T cells is acquired by mitochondrial transfer from mesenchymal stem cells

BackgroundApoptosis, a form of programmed cell death, is critical for the development and homeostasis of the immune system. Chimeric antigen receptor T (CAR-T) cell therapy, approved for hematologic cancers, retains several limitations and challenges associated with ex vivo manipulation, including CAR T-cell susceptibility to apoptosis. Therefore, strategies to improve T-cell survival and persistence are required. Mesenchymal stem/stromal cells (MSCs) exhibit immunoregulatory and tissue-restoring potential. We have previously shown that the transfer of umbilical cord MSC (UC-MSC)-derived mitochondrial (MitoT) prompts the genetic reprogramming of CD3+ T cells towards a Treg cell lineage. The potency of T cells plays an important role in effective immunotherapy, underscoring the need for improving their metabolic fitness. In the present work, we evaluate the effect of MitoT on apoptotis of native T lymphocytes and engineered CAR-T cells.MethodsWe used a cell-free approach using artificial MitoT (Mitoception) of UC-MSC derived MT to peripheral blood mononuclear cells (PBMCs) followed by RNA-seq analysis of CD3+ MitoTpos and MitoTneg sorted cells. Target cell apoptosis was induced with Staurosporine (STS), and cell viability was evaluated with Annexin V/7AAD and TUNEL assays. Changes in apoptotic regulators were assessed by flow cytometry, western blot, and qRT-PCR. The effect of MitoT on 19BBz CAR T-cell apoptosis in response to electroporation with a non-viral transposon-based vector was assessed with Annexin V/7AAD.ResultsGene expression related to apoptosis, cell death and/or responses to different stimuli was modified in CD3+ T cells after Mitoception. CD3+MitoTpos cells were resistant to STS-induced apoptosis compared to MitoTneg cells, showing a decreased percentage in apoptotic T cells as well as in TUNEL+ cells. Additionally, MitoT prevented the STS-induced collapse of the mitochondrial membrane potential (MMP) levels, decreased caspase-3 cleavage, increased BCL2 transcript levels and BCL-2-related BARD1 expression in FACS-sorted CD3+ T cells. Furthermore, UC-MSC-derived MitoT reduced both early and late apoptosis in CAR-T cells following electroporation, and exhibited an increasing trend in cytotoxic activity levels.ConclusionsArtificial MitoT prevents STS-induced apoptosis of human CD3+ T cells by interfering with the caspase pathway. Furthermore, we observed that MitoT confers protection to apoptosis induced by electroporation in MitoTpos CAR T-engineered cells, potentially improving their metabolic fitness and resistance to environmental stress. These results widen the physiological perspective of organelle-based therapies in immune conditions while offering potential avenues to enhance CAR-T treatment outcomes where their viability is compromised.

Clinical and prognostic significance of follicular helper and regulatory T cells in bladder cancer draining lymph nodes

Follicular helper and regulatory T cells (Tfh/TFR) cells are distinct subsets of CD4+ cells that have been recognized for their critical role in regulating cellular reactions within the germinal centers of lymphoid follicles. In the present study, we aimed to determine the presence and the frequency of these cells in draining lymph nodes of patients with bladder cancer (BC). Forty-six patients with BC who had undergone radical cystectomy and pelvic lymph node dissection were enrolled. Following routine pathological examination, a portion of the dissected lymph nodes was minced to obtain a single-cell suspension. Mononuclear cells were then separated using Ficoll-Hypaque gradient centrifugation, and the samples with proper viability (> 95%) were subjected to further analysis. To phenotype the follicular subsets, cells were stained with appropriate fluorochrome-conjugated antibodies specific for CD4, CXCR5, BCL6, and FOXP3. The cells were then acquired on a four-color flow cytometer. The data were analyzed with the FlowJo software version 10.8.1 package. Our analysis indicated that, on average 37.89 ± 16.36% of CD4+ lymphocytes in draining lymph nodes of patients with BC expressed CXCR5. The majority of them were negative for FOXP3, representing helper subsets (28.73 ± 13.66). A small percent simultaneously expressed BCL6 transcription factor (1.65% ± 1.35), designated as Tfh (CD4+BCL6+CXCR5+FOXP3-). While less than 10% of CD4+ lymphocytes expressed CXCR5 and FOXP3, 1.78 ± 2.54 were also positive for BCL6, known as TFR. Statistical analysis revealed that the frequency of both Tfh and TFR cells was higher in draining lymph nodes of patients with tumor-infiltrated nodes (P = 0.035 and P = 0.079, respectively) compared to those with negative ones. The percentage of these cells was also higher in high-grade tumors compared to low-grade ones (P = 0.031 for both). Our data collectively indicated that however approximately one third of CD4+ lymphocytes expressed CXCR5 and accordingly had the capacity to enter the follicles, less than 2% of them represented Tfh and TFR phenotypes. The percentage of these cells increased in progressed tumors and showed an association with negative prognostic factors.

Antioxidant Defenses, Oxidative Stress Responses, and Apoptosis Modulation in Spontaneous Abortion: An Immunohistochemistry Analysis of First-Trimester Chorionic Villi.

Oxidative stress (OS) and apoptosis are critical factors in placental development and function. Their interplay influences trophoblast proliferation, differentiation, and invasion, as well as vascular development. An imbalance between these processes can lead to pregnancy-related disorders such as preeclampsia, intrauterine growth restriction, and even spontaneous abortion. Our study seeks to elucidate the associations between preventive antioxidant/protective OS response factors-glutathione (GSH), MutT Homolog 1 (MTH1), and apoptotic regulation modulators-tumor protein p53 and B-cell lymphoma (Bcl-2) transcripts, in the context of spontaneous abortion (30 samples) versus elective termination of pregnancy (20 samples), using immunohistochemistry (IHC) to determine their proteomic expression in chorionic villi within abortive fetal placenta tissue samples. Herein, comparative statistical analyses revealed that both OS response factors, GSH and MTH1, were significantly under-expressed in spontaneous abortion cases as compared to elective. Conversely, for apoptotic regulators, p53 expression was significantly higher in spontaneous abortion cases, whereas Bcl-2 expression was significantly lower in spontaneous abortion cases. These findings suggest that a strong pro-apoptotic signal is prevalent within spontaneous abortion samples, alongside reduced anti-apoptotic protection, depleted antioxidant defenses and compromised oxidative DNA damage prevention/repair, as compared to elective abortion controls. Herein, our hypothesis that OS and apoptosis are closely linked processes contributing to placental dysfunction and spontaneous abortion was thus seemingly corroborated. Our results further highlight the importance of maintaining redox homeostasis and apoptotic regulation for a successful pregnancy. Understanding the mechanisms underlying this interplay is essential for developing potential therapies to manage OS, promote placentation, and avoid unwanted apoptosis, ultimately improving pregnancy outcomes. Antioxidant supplementation, modulation of p53 activity, and the enhancement of DNA repair mechanisms may represent potential approaches to mitigate OS and apoptosis in the placenta. Further research is needed to explore these strategies and their efficacy in preventing spontaneous abortion.

Vitamin C Improves Oocyte In Vitro Maturation and Potentially Changes Embryo Quality in Cattle.

To obtain high-quality bovine oocytes, the effects of vitamin C (VC) on the IVM of bovine oocytes and early embryo development were investigated. The results showed the following. (1) The IVM medium containing 50 µg/mL VC improved the oocyte maturation rate but did not affect the parthenogenetic embryo development. (2) The IVC medium containing 20 µg/mL VC improved the cleavage rate of the IVF embryos and enhanced the mRNA transcriptions of pluripotency gene Oct4, Sox2, Cdx2, and Nanog in the blastocysts but had no effects on the blastocyst rate. (3) Combining supplementation of 50 µg/mL VC in IVM medium + 20 µg/mL VC in IVC medium (named as VC 50/20, similar hereinafter) elevated the cleavage rate of IVF embryos and enhanced the mRNA expressions of Oct4, Sox2, Cdx2, and Nanog in the blastocysts. (4) Combination of VC 0/20 and VC 50/20 enhanced the transcription of anti-apoptotic gene Bcl-2 and VC 50/0 weakened the transcription of pro-apoptotic gene Bax, while VC 0/40 and VC 0/60 increased Bax expression and diminished the Bcl-2/Bax ratio in blastocysts. Together, employing 50 µg/mL VC improves the IVM of bovine oocytes and combination of VC 50/20 potentially changes bovine embryo quality by enhancing the expressions of the pluripotency genes and regulating the expressions of apoptosis-related genes.

Genetic modification of primary human B cells to model the process of B cell development in germinal centers

The main stages of maturation of antigen-specific B cells occur in the germinal centers of the lymph nodes. During the process of differentiation, a decision is made on which path the B cells will take to develop further. They will either turn into short-lived plasmablasts or memory B cells or plasma cells. The relationship between these processes is very important for the development of a productive humoral immune response. The goal of the work was to create a system that is capable of simulating ex vivo processes occurring in germinal centers. We used primary B cells from human peripheral blood as starting material. B lymphocytes were stimulated in vitro using feeder cells carrying CD40L molecules and recombinant IL-21. Upon IL-21/CD40L stimulation, B lymphocytes changed their morphology, surface phenotype, and functional activity. After active expansion for 10 days, further cell growth stopped, and after some time they died. To generate stably proliferating B cells, we used lentiviral transduction of IL-21/CD40L stimulated IgM+ B cells. For this purpose, lentivirus preparations were obtained that carried a cassette consisting of the BCL6 and BCL2L1 genes, separated by a sequence encoding the self-cutting peptide P2A, as well as a GFP reporter gene separated from the target genes by an IRES element. The cassette used ensured the synthesis of the Bcl-6 transcription factor and the Bcl-XL protein in target cells. The Bcl-6 repressor prevented B cells from undergoing terminal differentiation and becoming plasma cells, and the Bcl-XL protein had an anti-apoptotic effect. Transduced B cells proliferated for more than a month and maintained a plasmablast phenotype. Forty-two days after the start of stimulation, transduced B cells remained GFP-positive, coexpressed CD27 and CD38 antigens, carried surface CD20 and IgM, intracellular Bcl-6, Bcl-XL and IgM, retained IgM secretion, but remained negative for surface and intracellular IgG. The proven stimulation system will allow us to simulate key aspects of B cell development in germinal centers to study the formation of B cell memory, which will ultimately facilitate the development of effective vaccines.

FOXM1 c.1205C > A mutation is associated with unilateral Moyamoya disease and inhibits angiogenesis in human brain endothelial cells.

Unilateral moyamoya disease (MMD) represents a distinct subtype characterised by occlusive changes in the circle of Willis and abnormal vascular network formation. However, the aetiology and pathogenesis of unilateral MMD remain unclear. In this study, genetic screening of a family with unilateral MMD using whole-genome sequencing helped identify the c.1205C > A variant of FOXM1, which encodes the transcription factor FOXM1 and plays a crucial role in angiogenesis and cell proliferation, as a susceptibility gene mutation. We demonstrated that this mutation significantly attenuated the proangiogenic effects of FOXM1 in human brain endothelial cells, leading to reduced proliferation, migration, and tube formation. Furthermore, FOXM1 c.1205C > A results in increased apoptosis of human brain endothelial cells, mediated by the downregulation of the transcription of the apoptosis-inhibiting protein BCL2. These results suggest a potential role for the FOXM1 c.1205C > A mutation in the pathogenesis of unilateral MMD and may contribute to the understanding and treatment of this condition.

Abstract PO-010: Trial in Progress: Phase 1 Study of ARV-393, a PROTAC BCL6 Degrader, in Advanced Non-Hodgkin Lymphoma

Abstract Background: Non-Hodgkin lymphoma (NHL) represents a biologically and clinically diverse group of hematologic malignancies originating from B cells, T cells, and/or natural killer cells, with those of B-cell origin constituting ∼80–85% of all NHL cases. The BCL6 transcription factor is a key oncogenic driver of B-cell lymphomagenesis, and deregulated BCL6 expression is a common feature of diffuse large B-cell lymphoma (DLBCL), the most common type of NHL. BCL6 translocations have been observed in follicular lymphomas, the second most prevalent NHL, with studies suggesting that these translocations may portend a higher risk of transformation into aggressive lymphoma. BCL6 is also a lineage-defining transcription factor of T follicular helper cells, the cell of origin for angioimmunoblastic T-cell lymphoma (AITL). Moreover, BCL6 expression has been identified in biopsies of patients with AITL, and its continued expression was required for tumor growth in a mouse model of AITL, supporting BCL6 as a cancer driver in this type of NHL as well. ARV-393, an orally administered PROteolysis TArgeting Chimera (PROTAC) BCL6 degrader, is a bifunctional small molecule consisting of a BCL6-binding domain attached by a linker to a cereblon E3 ubiquitin ligase–binding domain. ARV-393 induces ubiquitination and subsequent degradation of BCL6 by the proteasome. In preclinical studies, ARV-393 induced rapid and robust degradation (>90%) of BCL6 in NHL cell lines and demonstrated dose-responsive tumor growth inhibition leading to tumor stasis or regression in xenograft models, supporting further investigation in patients with NHL. This multicenter, first-in-human, phase 1 study will evaluate the safety, tolerability, pharmacokinetics, pharmacodynamics, and preliminary antitumor activity of ARV-393 in patients with advanced B-cell NHL or AITL. Methods: Eligible patients (aged ≥18 years) have relapsed/refractory mature B-cell NHL and ≥2 prior systemic therapies, or histologically confirmed AITL that has recurred or progressed following institutional standard-of-care therapy. Patients must also have ≥1 measurable lesion at study entry, Eastern Cooperative Oncology Group performance status of 0 or 1, freshly biopsied or archival tumor tissue available, adequate organ function, no active central nervous system involvement, and no prior allogeneic stem cell transplant or solid organ transplantation. Autologous stem cell transplant must not have occurred ≤100 days and previous CAR T-cell therapy ≤60 days prior to cycle 1 day 1 of ARV-393 treatment. ARV-393 will be administered orally in 28-day cycles with dose escalation according to a Bayesian optimal interval design. The primary objectives are to evaluate the safety and tolerability of ARV-393, determine maximum tolerated dose if necessary, and identify the recommended phase 2 dose(s) and dosing schedule. Secondary objectives are to characterize the pharmacokinetic profile of ARV-393 and evaluate its preliminary anti-tumor activity. Enrollment for this study will open in 2024. Citation Format: Paolo Caimi, Scott Huntington, Sean Landrette, Sheryl M Gough, Vivian Dai, Bryan Jackson, Juan Chavez, Sarah Tannenbaum-Dvir, Matthew Matasar. Trial in Progress: Phase 1 Study of ARV-393, a PROTAC BCL6 Degrader, in Advanced Non-Hodgkin Lymphoma [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-010.

Abstract ND05: The discovery of ARV-393, a potent, orally bioavailable BCL6 targeting PROTAC® for the treatment of Non-Hodgkin’s Lymphoma

Abstract Discovery of ARV-393, a potent, orally bioavailable BCL6 targeting PROTAC® for the treatment of Non-Hodgkin’s Lymphoma (NHL)Dan Sherman, Sheryl M. Gough, Lynn DeCarr, Sarah Eaton, Mark Bookbinder, Jennifer Pizzano, Elizabeth Bortolon, John Corradi, Rashaun Wilson, Daniel Rogoz, Ram Lingamaneni, Wei Zhang, Michelle Zhang, Xin Chen, Gan Wang, Hanqing Dong, Michael Berlin, Keith R. Hornberger, Lawrence Snyder, Achal Pashine and Ian Taylor. Arvinas, Inc., 5 Science Park, New Haven, CT, USA. Approximately 18,000 cases of Diffuse large B-cell lymphomas (DLBCL) are diagnosed worldwide per year making this cancer the most prevalent (30%) of the non-Hodgkin’s Lymphomas (NHL). Despite the success of R-CHOP treatment, approximately 40% of patients are refractory or relapse. While much progress has been made with CAR-Ts and bi-specific antibodies following frontline therapy in recent years, there remains a need for an orally bioavailable small-molecule that targets molecular drivers of NHL. The BCL6 transcription factor has long been known as an oncogenic driver of B-cell malignancy. The BCL6 gene has been found to be translocated and/or mutated resulting in deregulation of its expression, predominantly in DLBCL and Burkitt’s lymphoma (BL). The deregulated expression and scaffolding role of BCL6 in the transcriptional repressor complex make it an ideal target for the PROteolysis Targeting Chimera (PROTAC®) technology with one BCL6 degrader recently entering Phase 1 trials. We undertook a medicinal chemistry campaign to identify a BCL6-targeting PROTAC®. This effort identified ARV-393, an orally bioavailable PROTAC® that recruits BCL6 and the E3 ligase cereblon to rapidly degrade BCL6 via the cell’s natural ubiquitin proteasome system. ARV-393 is potent, demonstrating DC50 and GI50 values <1 nM in numerous DLBCL and BL cell lines. This agent demonstrates deep BCL6 degradation in vitro and in vivo, resulting in excellent TGI in several tumor xenograft models supporting a first-in-human trial in 1H 2024. We will present the chemical structure of ARV-393 and selected pre-clinical data. Citation Format: Dan Sherman. The discovery of ARV-393, a potent, orally bioavailable BCL6 targeting PROTAC® for the treatment of Non-Hodgkin’s Lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr ND05.

Blastocoel fluid aspiration improves vitrification outcomes and produces similar sexing results of in vitro-produced cattle embryos compared to microblade biopsy

The potential applications of in vitro-produced (IVP) cattle embryos are significantly enhanced when combined with genotype selection and cryopreservation techniques. While trophectoderm (TE) biopsies are frequently used for genotyping, cell-free DNA (cfDNA) found in blastocoele fluid (BF) arises as a less-invasive method. Moreover, the blastocoel collapse produced by BF aspiration could be beneficial for embryo cryotolerance. This study was conducted to test the BF as a source of cell free-DNA (cfDNA) and to compare the BF to the TE biopsy in terms of sexing efficiency/accuracy, embryo survival and gene expression after vitrification/warming. IVP day 7 expanded blastocysts were artificially collapsed by aspiration of BF (VIT-Collapsed) or biopsied (VIT-Biopsied). After sample collection, embryos were vitrified/warmed by the Cryotop method and individually cultured in vitro. Intact fresh non-vitrified and vitrified/warmed blastocysts served as Fresh Control and VIT-Control, respectively. After sex identification of BF or TE biopsies and the corresponding surviving embryos, amplification efficiency and sexing accuracy were assessed. There were no differences between the BF and TE biopsy samples in terms of sexing accuracy or efficiency. Although all vitrified groups showed lower post-warming re-expansion rates (p < 0.05), the blastocyst re-expansion rates in the VIT-Collapsed group were comparable to those in the Fresh Control group whereas biopsied blastocysts showed the lowest (p < 0.05) re-expansion rates. VIT-Collapsed blastocysts had hatching rates that were comparable to those of Fresh Control blastocysts but significantly higher than those of the other vitrification treatments. Proapoptotic gene BAX was overexpressed in VIT-Biopsied embryos, whereas BCL2 transcripts were more abundant in the VIT-Collapsed group. On the other hand, VIT-Biopsied embryos showed altered ATP1B1- and AQP3-mRNA levels. The analysis of the cfDNA present in the BF is an efficient, minimally invasive approach to sex IVP cattle embryos. Besides, the artificial collapse of blastocoel prior to vitrification resulted in higher re-expansion and hatching ability than when embryos were vitrified after being biopsied.

Long-term Survival in a Patient With Transformation of Waldenström's Macroglobulinemia into DLBCL.

Waldenström's macroglobulinemia (WM) is a rare slow-growing B-cell lymphoma that is characterized by lymphoplasmacytic bone marrow infiltration and the production of monoclonal immunoglobulin M (IgM) paraprotein. In 5-10% of patients, WM undergoes transformation into diffuse large B-cell lymphoma (DLBCL), which is more aggressive, with poor prognosis and a low survival rate. Α 69-year-old woman was diagnosed with WM in 2009. She received six cycles of chemoimmunotherapy and a remarkable remission was achieved. However, in 2013 the disease transformed into DLBCL. The patient received chemotherapy and after the completion of the first cycle of therapy, the disease was significantly minimized. At the end of the therapy, there was no evidence of disease, and the patient remains disease-free. The cytogenetic profile of the patient did not reveal expression of BCL2 apoptosis regulator, BCL6 transcription repressor, Epstein-Barr virus small RNA, syndecan 1 nor cyclin D1. According to a staging system based on the platelet count, lactate dehydrogenase and previous treatment for WM, the described patient was classified as being at intermediate risk with an expected 2-year survival probability of 47% after WM transformation into DLBCL. However, the patient unexpectedly exceeded these prognostic indications. The findings for this patient are of great interest compared with the existing literature which suggests that the survival and prognosis for patients with transformed DLBCL are not favorable.

Effects of acute hypoxia and reoxygenation on histological structure, antioxidant response, and apoptosis in razor clam Sinonovacula constricta

Hypoxia is one of the major environmental problems limiting the healthy development of intensive aquaculture. Marine benthic shellfish are encountering heightened problems related to hypoxic stress as a result of ongoing human activities and aquaculture operations. Razor clam Sinonovacula constricta, a commercially valuable shellfish, has not yet been reported in studies on physiological changes caused by hypoxia and reoxygenation. To understand the negative effects of hypoxia and reoxygenation on the clams, we set up two low-oxygen concentration groups (DO 2.0 mg/L and DO 0.5 mg/L) and assessed multiple aspects of oxidative damage to their hepatopancreas and gills. After the hypoxic stress, the two tissues of the razor clam suffered varying degrees of damage, including cell degeneration and disruption of mitochondrial cristae. After reoxygenation, the 2.0 mg/L group recovered substantially, but the clams in the 0.5 mg/L group still unrecovered. The activities of antioxidant enzymes (MDA, T-AOC, SOD, GPX, and CAT) in clams were considerably altered by acute hypoxia and reoxygenation. Briefly, there was a growing and then declining trend in MDA, T-AOC, and SOD activities in the hepatopancreas, whereas GPX and CAT activities showed the converse trend. In the hepatopancreas and gills, the level of anti-apoptotic gene Bcl-2 transcripts gradually decreased with the duration of hypoxia and increased following reoxygenation. However, changes in the transcript level of the pro-apoptotic gene Bax were in contrast to that of Bcl-2. The TUNEL assay revealed that hypoxia caused apoptosis. Furthermore, at DO 0.5 mg/L, the degree of apoptosis was more significant than at DO 2.0 mg/L, and hepatopancreatic apoptosis was more severe than gill apoptosis. Collectively, our findings imply that hypoxia induces oxidative stress, histological damage, and apoptosis in razor clams in a concentration-dependent and tissue-specific manner. These consequences serve as a reminder that prolonged recovery periods may be required for razor clams to fully recover from oxidative damage resulting from hypoxia-reoxygenation episodes.

The TRIM21-FOXD1-BCL-2 axis underlies hyperglycaemic cell death and diabetic tissue damage

Chronic hyperglycaemia is a devastating factor that causes diabetes-induced damage to the retina and kidney. However, the precise mechanism by which hyperglycaemia drives apoptotic cell death is incompletely known. Herein, we found that FOXD1, a FOX family transcription factor specifically expressed in the retina and kidney, regulated the transcription of BCL-2, a master regulator of cell survival. Intriguingly, the protein level of FOXD1, which responded negatively to hyperglycaemic conditions, was controlled by the TRIM21-mediated K48-linked polyubiquitination and subsequent proteasomal degradation. The TRIM21-FOXD1-BCL-2 signalling axis was notably active during diabetes-induced damage to murine retinal and renal tissues. Furthermore, we found that tartary buckwheat flavonoids effectively reversed the downregulation of FOXD1 protein expression and thus restored BCL-2 expression and facilitated the survival of retinal and renal tissues. In summary, we identified a transcription factor responsible for BCL-2 expression, a signalling axis (TRM21-FOXD1-BCL-2) underlying hyperglycaemia-triggered apoptosis, and a potential treatment for deleterious diabetic complications.

Melatonin exerts neuroprotective effects in mice with spinal cord injury by activating the Nrf2/Keap1 signaling pathway via the MT2 receptor.

Spinal cord injury (SCI) is a devastating event that often leads to severe disability, and effective treatments for SCI are currently limited. The present study investigated the potential effects and specific mechanisms of melatonin treatment in SCI. Mice were divided into Sham (Sham), Vehicle (Veh), Melatonin (Mel), and Melatonin + 4-phenyl-2-propionamidotetralin (4P-PDOT) (Mel + 4PP) groups based on randomized allocation. The expression of MT2 and the nuclear factor-erythroid 2-related factor 2 (Nrf2)/Keap1 signaling pathways were examined, along with oxidative stress indicators, inflammatory factors and GFAP-positive cells near the injury site. The polarization of microglial cells in different inflammatory microenvironments was also observed. Cell survival, motor function recovery and spinal cord tissue morphology were assessed using staining and Basso Mouse Scale scores. On day 7 after SCI, the results revealed that melatonin treatment increased MT2 protein expression and activated the Nrf2/Keap1 signaling pathway. It also reduced GFAP-positive cells, mitigated oxidative stress, and suppressed inflammatory responses around the injury site. Furthermore, melatonin treatment promoted the polarization of microglia toward the M2 type, increased the number of neutrophil-positive cells, and modulated the transcription of Bax and Bcl2 in the injured spinal cord. Melatonin treatment alleviated the severity of spinal injuries and facilitated functional recovery in mice with SCI. Notably, blocking MT2 with 4P-PDOT partially reversed the neuroprotective effects of melatonin in SCI, indicating that the activation of the MT2/Nrf2/Keap1 signaling pathway contributes to the neuroprotective properties of melatonin in SCI. The therapeutic and translational potentials of melatonin in SCI warrant further investigation.

Acute Myeloid Leukemia Driven IL-3 Dependent Upregulation of BCL-2 in Non-Malignant Hematopoietic Progenitor Cells Increases Venetoclax Induced Cytopenias

The BH3 mimetic venetoclax, in combination with low dose cytarabine, decitabine or azacitidine has shown clinical efficacy in newly diagnosed acute myeloid leukemia (AML) (1, 2). This has been a significant advance, particularly in the treatment of older AML patients, and those who are ineligible for intensive chemotherapy, who historically have been difficult to treat. Venetoclax selectively inhibits the BCL-2 protein which is overexpressed in AML in order to activate intrinsic apoptosis. However, this treatment regime is associated with cytopenias including neutropenia, febrile neutropenia, and thrombocytopenia which leaves patients immunocompromised (3). The underlying cause of cytopenias in the context of venetoclax-treated AML remains unexplained. Here we describe a mechanism for venetoclax-induced cytopenias in AML. Two established AML models were used to assess BCL-2 expression profile in HPCs. Isolated LSK cells were co-cultured with mouse AML (HOXA9/Meis1 or MN1 overexpressing cells). RT-qPCR analysis identified BCL-2 upregulation in LSKs co-cultured with both AML cell types. To understand the significance of this in vivo, MN1 cells were engrafted into C57BL/6 mice and LSKs were FACS purified. Analysis confirmed increased BCL-2 gene and protein expression by RT-qPCR and flow cytometry when compared to control LSKs. Next, to recapitulate the clinical cytopenias, MN1 engrafted mice or control mice were treated with venetoclax for 7 days by oral gavage. Flow cytometry analysis of bloods and bone marrow in venetoclax-treated AML mice determined neutrophil depletion and reduced numbers of HPCs, namely LSK, MPP, HSC and GMP, compared to untreated AML mice . Todetermine the mechanism of BCL-2 upregulation in LSKs we explored various signalling pathways using targeted inhibitors on LSKs cultured with MN1 conditioned media. Using this method, we established that interleukin 3 (IL-3) activates BCL-2 transcription in LSKs leading to a dependency on pro-survival BCL-2 protein. Furthermore, treatment of LSKs, cultured in MN1 conditioned media with IL-3 neutralising antibody MP2-8F8 reduced BCL-2 gene and protein expression in LSKs. Finally, MN1 engrafted mice were treated with venetoclax and IL-3 neutralising antibody in combination which restored neutrophil levels to numbers observed in untreated AML mice. Here, we demonstrate that BCL-2 is overexpressed in non-malignant HPCs during AML progression using two AML models. In addition, using our syngeneic mouse model we show that venetoclax induces HPC depletion. In vitro studies confirm that IL-3 mediates BCL-2 upregulation in HPCs - this sensitises HPCs to venetoclax and causes cytopenias. We also demonstrate that neutrophils can be recovered using IL-3 neutralisation in combination with venetoclax in AML engrafted mice. Taken together, these findings provide biologic insight for IL-3 inhibition alongside venetoclax to reduce the incidence of cytopenias observed in elderly AML patients.

Exosomal circKIAA1797 Regulates Cell Progression and Glycolysis by Targeting miR-4429/PBX3 Pathway in Gastric Cancer.

In recent years, circular RNAs (circRNAs) are extensively studied in the progression of various types of cancer, while the mechanism of circKIAA1797 is rarely studied in gastric cancer (GC). Hence, this research aimed to investigate the expression of exosomal circKIAA1797 and its biological function in GC cells. Exosomes were extracted from the serum of GC patients and identified by transmission electron microscopy (TEM) and nanoparticle tracking analyzer (NTA). CD81, CD63, Bcl-2, Bax, and pre-leukemia transcription factor 3 (PBX3) protein levels were detected using western blot assay. circKIAA1797, microRNA-4429 (miR-4429), and PBX3 mRNA were determined by quantitative real-time PCR (RT-qPCR). Cell proliferation, migration, invasion, and apoptosis were assessed using colony formation assay, 5-Ethynyl-2'-deoxyuridine (EdU) assay, transwell assay, and flow cytometry assay. Glucose consumption and lactate production levels were examined using glycolysis detection kits. The interaction between miR-4429 and circKIAA1797 or PBX3 was identified using dual-luciferase reporter assay, RNA pull-down assay, and RNA immunoprecipitation (RIP) assay. Xenograft mouse model assay was used to investigate the effect of exosomal circKIAA1797 in vivo. It was found that circKIAA1797 was up-regulated in GC tissues and cells, as well as in the exosomes derived from the serum of GC patients. Silencing of exosomal circKIAA1797 could hamper cell progression and glycolytic metabolism of GC. Mechanically, circKIAA1797 acted as a sponge of miR-4429 to regulate PBX3 expression. Moreover, the knockdown of exosomal circKIAA1797 repressed tumor growth in vivo. Our data demonstrated that knockdown of exosomal circKIAA1797 suppressed GC malignant phenotypes by regulating miR-4429/PBX3 axis, which might offer a promising therapeutic strategy for GC treatment.

Potent anti-cancer activity of Sphaerocoryne affinis fruit against cervical cancer HeLa cells via inhibition of cell proliferation and induction of apoptosis

BackgroundCervical cancer remains a significant global health issue, highlighting the need for effective therapeutic strategies. Given that Sphaerocoryne affinis (SA) has shown potential anti-cancer activity in several cancer types, herein, we investigate the effects of SA fruit (SAF) on human cervical cancer HeLa cells and their underlying mechanisms of action.MethodsSAF extract cytotoxicity was assessed in various cancer cell lines. The effects of the hexane fraction (SAF-Hex) on HeLa cell viability, cell cycle protein expression, apoptosis, and DNA damage were evaluated using cytotoxicity assays, Western blotting, quantitative PCR, 4′,6-diamidino-2-phenylindole (DAPI) staining, and a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay.ResultsSAF-Hex selectively inhibited HeLa cell viability with an IC50 of 4.20 ± 0.36 µg/mL and a selectivity index of 5.11 ± 0.58. The time-dependent cytotoxicity assay showed decreased cell survival after 48 h of treatment, accompanied by morphological changes and apoptotic bodies in HeLa cells. SAF-Hex also suppressed HeLa cell cycle proteins (Cyclin E, CDK2, and CDK1), reduced PCNA transcription, and diminished AKT and mTOR activation, thus inhibiting cell proliferation. The increased γH2AX expression, DNA fragmentation, and caspases-3 and -9 activation indicated SAF-Hex-induced DNA damage and apoptosis. However, the BAX/BCL-2 ratio remained unchanged, and BAX and BCL2 expression was attenuated.ConclusionSAF-Hex effectively inhibits HeLa cell proliferation and induces DNA damage in that cervical cancer cell line activating apoptosis through the intrinsic pathway. Interestingly, the BAX/BCL-2 ratio remained unchanged while BAX and BCL2 transcription was attenuated. Hence, further research is required to explore this unexpected finding and facilitate the development of novel therapies targeting cervical cancer HeLa cells.

Difenoconazole disrupts carp intestinal physical barrier and causes inflammatory response via triggering oxidative stress and apoptosis

As a common fungicide, difenoconazole (DFZ) is widespread in the natural environment and poses many potential threats. Carp makes up a significant proportion of China's freshwater aquaculture population and are vulnerable to the DFZ. Therefore, this study investigated the effects of DFZ (0.488 mg/L and 1.953 mg/L) exposure for 4 d on the intestinal tissues of carp and explored the mechanisms. Specifically, DFZ exposure caused pathological damage to the intestinal tissues of carp, reducing the expression levels of intestinal tight junction proteins, and leading to damage to the intestinal barrier. In addition, DFZ exposure activated the NF-κB signaling pathway, increasing the levels of pro-inflammatory factors (TNF-α, IL-1β, IL-6) and decreasing the levels of anti-inflammatory factors (IL-10, TGF-β1). As disruption of the intestinal barrier is closely linked to oxidative stress and apoptosis, we have conducted research in both areas for this reason. The results showed that DFZ exposure elevated reactive oxygen species in carp intestines, decreased antioxidant enzyme activity, and suppressed the expression of oxidative stress-related genes. TUNEL results showed that DFZ induced the onset of apoptosis. In addition, the expression levels of apoptosis-related genes and proteins were examined. Western blotting results showed that DFZ could upregulate the protein expression levels of Bax, Cytochrome C and downregulate the protein levels of Bcl-2. qPCR results showed that DFZ could upregulate the transcript levels of Bax, Caspase-3, Caspase-8 and Caspase-9 and downregulate the transcript levels of Bcl-2 transcript levels. This suggests that DFZ can induce apoptosis of mitochondrial pathway in carp intestine. In conclusion, DFZ can induce oxidative stress and apoptosis in carp intestine, leading to the destruction of intestinal physical barrier and the occurrence of inflammation. Our data support the idea that oxidative stress and apoptosis are important triggers of pesticide-induced inflammatory bowel illness.

Apoptotic Changes, Oxidative Stress and Immunomodulatory Effects in the Liver of Japanese Seabass (Lateolabrax japonicus) Induced by Ammonia-Nitrogen Stress during Keep-Live Transport.

This study investigated the effects of NH3-N on antioxidant responses, histoarchitecture, and immunity of Japanese seabass (Lateolabrax japonicus) during keep-live transport. The findings suggest that NH3-N stress transport alters the transcription of P53, Caspase 9, Bcl2, Caspase 3 and Bax genes, demonstrating that NH3-N stress can trigger the apoptotic pathway of P53-Bax-Bcl2 and Caspase and induce apoptosis. NH3-N stress transport also evoked transcriptional upregulation of inflammatory cytokines (tumor necrosis factor α (TNF-α), Toll-like receptor 3 (TLR-3), nuclear factor kappa β (NF-κB), interleukin 6 (IL-6) and interleukin 1β (IL-1β)) and increased complement C3, C4, lysozyme (LZM) and immunoglobulin (IgM) levels, activating the innate immunological system during keep-live transport. In addition, NH3-N stress transport altered changes in the levels of superoxide dismutase (SOD), catalase (CAT), glutathione-related enzymes, and heat shock proteins 70 and 90 in the liver, indicating that the antioxidant system and Hsp protected the cells from NH3-N-induced oxidative stress. When excess ROS were not removed, they caused the body to respond with immunological and inflammatory responses, as well as apoptosis and tissue damage. This helps towards understanding the effect of NH3-N levels on sea bass during keep-live transport.

Tissue-Specific Vulnerability to Apoptosis in Machado-Joseph Disease.

Machado-Joseph disease (MJD) is a dominant neurodegenerative disease caused by an expanded CAG repeat in the ATXN3 gene encoding the ataxin-3 protein. Several cellular processes, including transcription and apoptosis, are disrupted in MJD. To gain further insights into the extent of dysregulation of mitochondrial apoptosis in MJD and to evaluate if expression alterations of specific apoptosis genes/proteins can be used as transcriptional biomarkers of disease, the expression levels of BCL2, BAX and TP53 and the BCL2/BAX ratio (an indicator of susceptibility to apoptosis) were assessed in blood and post-mortem brain samples from MJD subjects and MJD transgenic mice and controls. While patients show reduced levels of blood BCL2 transcripts, this measurement displays low accuracy to discriminate patients from matched controls. However, increased levels of blood BAX transcripts and decreased BCL2/BAX ratio are associated with earlier onset of disease, indicating a possible association with MJD pathogenesis. Post-mortem MJD brains show increased BCL2/BAX transcript ratio in the dentate cerebellar nucleus (DCN) and increased BCL2/BAX insoluble protein ratio in the DCN and pons, suggesting that in these regions, severely affected by degeneration in MJD, cells show signs of apoptosis resistance. Interestingly, a follow-up study of 18 patients further shows that blood BCL2 and TP53 transcript levels increase over time in MJD patients. Furthermore, while the similar levels of blood BCL2, BAX, and TP53 transcripts observed in preclinical subjects and controls is mimicked by pre-symptomatic MJD mice, the expression profile of these genes in patient brains is partially replicated by symptomatic MJD mice. Globally, our findings indicate that there is tissue-specific vulnerability to apoptosis in MJD subjects and that this tissue-dependent behavior is partially replicated in a MJD mouse model.

Abstract A18: STC-15, a novel METTL3 inhibitor, and its combination with Venetoclax confer anti-tumour activity in AML models

Abstract N6-methyladenosine (m6A) is one of the most abundant RNA modifications, which influences mRNA and lncRNA localization, half-life, translation, and splicing. The majority of m6A modifications on cellular mRNAs are deposited by the RNA methyltransferase METTL3. To date, METTL3 has been implicated in the initiation and progression of multiple cancer types, with the highest expression of METTL3 mRNA observed in acute myeloid leukemia (AML). Currently, one line of standard of care therapy for AML patients is Venetoclax, which targets the anti-apoptotic protein BCL2. It was shown that m6A, deposited by METTL3 on BCL2 transcript, affects BCL2 mRNA stability and translation. Storm Therapeutics has developed potent and selective METTL3 inhibitors, including the clinical candidate STC-15. Here, we explore pharmacological inhibition of METTL3 as monotherapy or in combination with Venetoclax in AML models in vitro and in vivo. Sulforhodamine B and CellTiterGloTM assays were used to assess the viability of AML cell lines and patient-derived xenografts (PDXs), respectively, following METTL3 inhibition in vitro. BCL2 protein level was evaluated by Western blotting. SynergyFinder software was used to assess the degree of synergy between METTL3 inhibitors and Venetoclax. Intra-tibial implantation of human-derived AML cells (AML-PDXs) in NSG mice was used to determine single agent and combination therapy efficacy. Multiple AML cells lines and AML-PDXs were sensitive to pharmacological inhibition of METTL3 in vitro, as assessed by loss of viability. Treatment with METTL3 inhibitors led to downregulation of BCL2 protein level in several AML cell lines, as previously suggested by literature. Based on these results, the synergy between METTL3 inhibition and Venetoclax was assessed. Matrix-combination experiments have shown a high degree of synergy between the two drugs (defined by a synergy score &amp;gt;10) in THP-1 and MOLM-13 cell lines. To test METTL3 inhibition as a monotherapy and in combination with Venetoclax in vivo, three AML-PDX studies were initiated. Significantly lower spleen weight was observed in all animals treated with STC-15 or STC-15 + Venetoclax, and reduced number of circulating hCD45+ cells was observed in 2 out of the 3 models. In one of the models, STC-15 monotherapy outperformed Venetoclax (median survival 68 days vs 58 days, respectively), while the combination therapy extended median group survival to 85 days in comparison to 51.5 days in the vehicle group. In conclusion, we demonstrated that METTL3 inhibition results in anti-tumour effects across different AML models. Moreover, we demonstrated a synergistic effect between the novel METTL3 inhibitor STC-15 and Venetoclax, both in vitro and in vivo. These studies provide evidence for the utility of METTL3 inhibitor as a new therapeutic agent to treat AML. Currently, STC-15 is under clinical development (NCT05584111). Citation Format: Lina Vasiliauskaite, Yaara Ofir-Rosenfeld, Mark Albertella, Coralie Hoareau-Aveilla, Jerry McMahon, Oliver Rausch. STC-15, a novel METTL3 inhibitor, and its combination with Venetoclax confer anti-tumour activity in AML models [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A18.